The objective of the proposed studies is to understand the protein-protein interactions involved in the assembly of the bacterial type III secretion needle apparatus. This apparatus resembles a syringe on the bacterial surface and is used by many pathogens to inject virulence factors into target cells to initiate human diseases. The needle-like assembly consists of about 120 copies of identical proteins that are arranged in a superhelical manner, and the apex of this needle is capped by tip proteins. Over the past two years, we have completed the NMR structures of two needle monomers: BsaL from Burkholderia pseudomallei, a pathogen associated with biowarfare, and PrgI from Salmonella typhimurium, a pathogen associated with food poisoning. We have also used NMR to identify the key residues involved in the binding interaction between the needle and tip proteins of Shigella flexneri. Our goal is to elucidate how the needle apparatus is assembled by determining the precise protein-protein contacts involving the needle and tip proteins. We will combine NMR with electron microscopy and molecular modeling to determine the structure of the assembled needle. Our aims are: (#1) determine how the needle is assembled, (#2) determine how the tip protein docks on the needle, and (#3) use mutagenesis and microbiological assays to correlate the structural results to needle assembly and virulence in vivo. Many pathogens rely on the needle apparatus to infect millions of people worldwide and many are potential agents of bioterrorism. Because this apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive approach for the development of novel anti-infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly. PUBLIC HEALTH RELEVANCE The appearance of many antibiotic resistant bacterial pathogens poses a major problem in public health and safety and many of these bacteria rely on a needle-like protein assembly to infect their hosts. The proposed research seeks to elucidate how this needle is assembled by determining the protein-protein interactions of its components. This knowledge will then be used in the design of novel anti-bacterial agents.